Project Summary (Duerfeldt Project) Clostridium difficile infection (CDI), is a leading cause of hospital-acquired illness in the United States (500,000 cases in 2011) and represents a unique challenge to therapeutic development, as it is both facilitated by and clinically managed with traditional antibiotics. The inability to effectively treat CDI produces ~$5B in excess healthcare costs annually, a number that will continue to rise if new drug targets and treatment strategies are not identified. As a key regulator of virulence in infectious bacteria, and given its roles in mediating protein turnover and bacterial homeostasis, caseinolytic protease P (ClpP) has emerged as a new target for antimicrobial development. Indeed, ClpP represents a unique target, as both ClpP inhibition and activation have therapeutic utility, with each strategy affecting different aspects of bacterial pathogenicity. This provides an opportunity to determine the therapeutic potential of two orthogonal strategies on a single target, a rare phenomenon in drug discovery. The ClpP system in C. difficile, however, has not been characterized, thus impeding its pursuit as a new target to treat CDI. Importantly, C. difficile is unique from typical gut microflora, in that it expresses two isoforms of ClpP (ClpP1 and ClpP2). Our preliminary data and analyses of existing structure-function profiles suggest that the ClpP system in C. difficile is not only unique from commensal organisms, but also distinct from any pathogenic system disclosed to date. We hypothesize that in C. difficile ClpP maintains its evolutionarily conserved role as a major pathogenic regulator but exhibits unique structural and functional characteristics that provide avenues for the development of selective therapeutic agents. A multi-dimensional approach is required to address the proposed initiatives and we have strategically assembled a collaborative network of researchers with expertise in C. difficile microbiology, protein chemistry, protein crystallization/crystallography, molecular biology, and medicinal chemistry. To test our hypothesis, we propose to 1) characterize the structurally unique aspects of apo- and modulator-bound cdClpP complexes; 2) define the specific roles of ClpP in C. difficile pathogenesis; and 3) integrate screening platforms to identify novel leads for cdClpP specific modulation. The research is significant because new drug targets are required to effectively treat CDI. We expect that our studies will uncover novel ClpP involvement in pathogenicity and reveal distinct structural and functional aspects of the ClpP system in C. difficile amenable to selective regulation. Additionally, our studies will provide novel ClpP inhibitors and activators that will enable medicinal chemistry campaigns towards the selective targeting of this proteolytic system as an anti-CDI strategy. Therefore, this research will significantly advance the basic understanding of a unique ClpP system and enable translationally focused initiatives.